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Abstract

Na-terminal acetylation (NTA) is one of the most common protein modifications in eukaryotes, targeting up to 80% of cytosolic proteins in humans and plants. It is catalyzed by Na-terminal acetyltransferases (Nats) called NatA-NatH, which transfer the acetyl group from AcetylCoA to the N-terminus of the substrate proteins. NatA is the major acetyltransferase in eukaryotes, targeting up to 50% of proteins in humans and Arabidopsis. In the latter, NTA by NatA is essential and stabilizes NatA substrates by masking a nonAc-X2/N-degron, which targets proteins for degradation by the ubiquitin proteasome system. The knockout of NatA leads to embryo lethality in A.thaliana, while NatA-depleted plants are strongly retarded in growth and suffer from enhanced protein turnover. However, they are more resistant to diverse stresses like pathogen attack and water limitation, which are sensed by the phytohormones salicylic acid (SA) and abscisic acid (ABA), respectively. The first project of this thesis aimed at identifying biological processes involved in the generation of the dwarf phenotype observed in NatA-depleted mutants. I identified six candidate dominant mutations reverting the growth retardation in NatA-depleted mutant muse6-1 in a forward genetic screen. One of them was a novel mutation in a pre-mRNA splicing machinery which restored the splicing of a mutated auxiliary subunit of NatA, NAA15, leading to increased NatA levels and restoration of the WT-like phenotype. The next part of this work focused on understanding the role of NatA in SA and ABA-triggered stress responses and dissecting the involvement of the enhanced protein turnover in NatAdepleted plants for growth retardation under non-stressed conditions and the enhanced resistance to protein-harming stresses, like water withdrawal. By genetic crossing of NatA-depleted plants with mutants impaired in ABA biosynthesis and signaling, I demonstrated that the drought resistance of NatA-depleted plants is caused by a constitutively activated ABA response upstream of OPEN STOMATA ONE 1 (OST1) but downstream of ABA biosynthesis and initial perception. The generation of crosses between WT Landsberg erecta and the NatA-depleted mutant, muse6-1, disentangled the role of the constitutively induced ABA response and upregulated protein turnover in the generation of the dwarf phenotype in NatA-depleted mutants. WT Ler;muse6-1 crosses partially rescued the phenotype of NatA-depleted mutants by restoration of WT-like protein turnover rate, albeit the stomata were still closed. While ABA mediates the stomata closure in NatA-depleted mutants, it does not contribute to the upregulated protein turnover, which is the major contributor to the dwarf phenotype of NatA-depleted mutants. This thesis disproved the role of the accumulation of yet another phytohormone, SA, in the generation of the dwarf phenotype in NatA-depleted mutants and the upregulation of autophagy in these lines. However, the higher internal levels of SA in NatA mutants prime the pathogen responses in these lines by upregulating the pathogen-response-related genes, yielding them resistant to H.a. Noco2. The depletion of NatA leads to the generation of unstable NatA substrates, which are turned over at a higher rate by the UPS. In this thesis, I focused on understanding the role of autophagy in NTA-mediated proteostasis. Various measurements of markers and substrates of autophagy provided evidence for its upregulation in NatA-depleted mutants and its role in the turnover of polyubiquitinated proteins generated upon NatA-depletion. Furthermore, I demonstrated that NatA-depleted mutants accumulate higher amounts of protein aggregates and that autophagy is important for surveillance mechanisms in these lines. In summary, this thesis highlights the significant role of NTA by NatA in regulating the global turnover of the proteome, particularly under biotic- and abiotic stresses.